Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Domžale, Slovenia.
Agricultural Institute of Slovenia, Ljubljana, Slovenia.
Anim Biotechnol. 2021 Jun;32(3):282-291. doi: 10.1080/10495398.2019.1687091. Epub 2019 Nov 18.
Skin-derived tissue cultures are a useful model to study molecular mechanisms of skin renewal and pathogenesis of dermal diseases. Horses often suffer from skin diseases, skin trauma and problems with proper wound healing, which could be improved by grown keratinocyte grafts. Herein we describe establishment and characterization of equine skin-derived primary cell cultures, using enzymatic and explant methods. The established cell lines of primary equine keratinocytes (peK) maintained high proliferative capacity for over five passages and expressed different epithelial/keratinocyte-specific markers. Characterization of the primary culture was performed in parallel with localization studies of the markers in the skin histological sections, using commercially available antibodies. Relative expression of typical differentiation stage-specific markers was determined in the established cell lines, using RT-qPCR. Basal (proliferating) keratinocytes were the predominant cell type in the established cell lines, but low expression of post-mitotic keratinocyte markers was also detected. Differences in marker expression were observed neither between the peK originating from two different animals nor between the peK established with two different methods (enzymatically or by explanting). The described methods in combination with the suggested characterization and differentiation markers are suitable for establishment of proliferating peK and evaluation of their differentiation status.
皮肤组织培养是研究皮肤更新的分子机制和皮肤疾病发病机制的有用模型。马经常患有皮肤疾病、皮肤创伤和伤口愈合不当的问题，而通过培养的角质形成细胞移植可以改善这些问题。本文描述了使用酶和外植体方法建立和表征马原代皮肤细胞培养物。建立的原代马角质形成细胞（peK）系保持了超过 5 代的高增殖能力，并表达了不同的上皮/角质形成细胞特异性标志物。通过使用商业可得的抗体在皮肤组织学切片中对标志物进行定位研究，对原代培养物进行了表征。使用 RT-qPCR 确定了在建立的细胞系中典型分化阶段特异性标志物的相对表达。在建立的细胞系中，基底（增殖）角质形成细胞是主要的细胞类型，但也检测到低水平的有丝分裂后角质形成细胞标志物的表达。在源自两个不同动物的 peK 之间，或在用两种不同方法（酶或外植体）建立的 peK 之间，均未观察到标志物表达的差异。描述的方法与建议的表征和分化标志物相结合，适用于增殖 peK 的建立和分化状态的评估。
